In 1533, at the age of fourteen, Caterina married Henry, second son of King Francis I and Queen Claude of France. Under the gallicised version of her name, Catherine de Médicis, she was Queen consort of France as the wife of King Henry II of France from 1547 to 1559. Throughout his reign, Henry excluded Catherine from participating in state affairs and instead showered favours on his chief mistress, Diane de Poitiers, who wielded much influence over him. Henry's death thrust Catherine into the political arena as mother of the frail fifteen-year-old King Francis II. When he died in 1560, she became regent on behalf of her ten-year-old son King Charles IX and was granted sweeping powers. After Charles died in 1574, Catherine played a key role in the reign of her third son, Henry III. He dispensed with her advice only in the last months of her life.

Catherine's three sons reigned in an age of almost constant civil and religious war in France. The problems facing the monarchy were complex and daunting. At first, Catherine compromised and made concessions to the rebelling Protestants, or Huguenots, as they became known. She failed, however, to grasp the theological issues that drove their movement. Later, she resorted in frustration and anger to hard-line policies against them. In return, she came to be blamed for the excessive persecutions carried out under her sons' rule, in particular for the St. Bartholomew's Day massacre of 1572, in which thousands of Huguenots were killed in Paris and throughout France.

Some historians have excused Catherine from blame for the worst decisions of the crown, though evidence for her ruthlessness can be found in her letters. In practice, her authority was always limited by the effects of the civil wars. Her policies, therefore, may be seen as desperate measures to keep the Valois monarchy on the throne at all costs, and her patronage of the arts as an attempt to glorify a monarchy whose prestige was in steep decline. Without Catherine, it is unlikely that her sons would have remained in power. The years in which they reigned have been called "the age of Catherine de' Medici". According to one of her biographers Mark Strage, Catherine was the most powerful woman in sixteenth-century Europe.